title: Gene orders in the upstream of 16S rRNA genes divide genera of the family Halobacteriaceae into two groups.
abstract: In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to concerted evolution. At the time of writing, the genus Haloarcula of the family Halobacteriaceae comprises nine species with validly published names, all of which possess two to four highly heterogeneous 16S rRNA genes. Existence of multiple heterogeneous 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequence data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous ones in the members of the family Halobacteriaceae. In this study, we found that the dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene in each of ten strains of the family Halobacteriaceae whose genome sequences have been determined, and the direction of the pyrD gene was opposite to that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD gene was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the tRNA-Ala gene or the 23S rRNA gene to determine the 16S rRNA gene sequences preceded by the pyrD gene, and to see if the pyrD gene is conserved in the immediate upstream of rRNA operon(s) in the type strains of the type species of 28 genera of the family Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4000 bp, while eleven type strains, including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I haloarchaea, originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I haloarchaea also revealed the absence of the pyrD gene in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of the family Halobacteriaceae, a pyrD gene transposition event happened in one group and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and 'the rest' of the present family Halobacteriaceae.
authors.affiliation: Bio-Nano Electronics Research Center, Toyo University, Kawagoe, Saitama, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Minegishi
authors.affiliation: Halophiles Research Institute, Noda, Chiba, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kamekura
authors.affiliation: Center for Gene Research, Nagoya University, Nagoya, Aichi, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kitajima-Ihara
authors.affiliation: Department of Chemistry and Environmental Technology, School of Engineering, Kinki University, Higashi-Hiroshima, Hiroshima, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Nakasone
authors.affiliation: Faculty of Science and Engineering, Toyo University, Kawagoe, Saitama, Japan.|Bio-Nano Electronics Research Center, Toyo University, Kawagoe, Saitama, Japan.
authors.forename: Akinobu
authors.initials: A
authors.lastname: Echigo
authors.affiliation: Graduate School of Interdisciplinary New Science, Toyo University, Kawagoe, Saitama, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shimane
authors.affiliation: Graduate School of Interdisciplinary New Science, Toyo University, Kawagoe, Saitama, Japan.|Faculty of Science and Engineering, Toyo University, Kawagoe, Saitama, Japan.|Bio-Nano Electronics Research Center, Toyo University, Kawagoe, Saitama, Japan.
authors.forename: Ron
authors.initials: R
authors.lastname: Usami
authors.affiliation: Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Itoh
authors.affiliation: Center for Gene Research, Nagoya University, Nagoya, Aichi, Japan.
authors.forename: Kunio
authors.initials: K
authors.lastname: Ihara
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004081:Dihydroorotate Oxidase / Q000235:genetics*; D006216:Halobacteriaceae / Q000145:classification* / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D004081:Dihydroorotate Oxidase